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1.
ChemMedChem ; 5(11): 1871-9, 2010 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-20677318

RESUMO

In an effort to identify broadly active inhibitors of HIV-1 entry into host cells, we previously reported a family of dodecamer triazole-peptide conjugates with nanomolar affinity for the viral surface protein gp120. This peptide class exhibits potent antiviral activity and the capacity to simultaneously inhibit interaction of the viral envelope protein with both CD4 and co-receptor. In this investigation, we minimized the structural complexity of the lead triazole inhibitor HNG-156 (peptide 1) to explore the limits of the pharmacophore that enables dual antagonism and to improve opportunities for peptidomimetic design. Truncations of both carboxy- and amino-terminal residues from the parent 12-residue peptide 1 were found to have minimal effects on both affinity and antiviral activity. In contrast, the central triazole(Pro)-Trp cluster at residues 6 and 7 with ferrocenyl-triazole(Pro) (Ftp) was found to be critical for bioactivity. Amino-terminal residues distal to the central triazole(Pro)-Trp sequence tolerated decreasing degrees of side chain variation upon approaching the central cluster. A peptide fragment containing residues 3-7 (Asn-Asn-Ile-Ftp-Trp) exhibited substantial direct binding affinity, antiviral potency, dual receptor site antagonism, and induction of gp120 structuring, all properties that define the functional signature of the parent compound 1. This active core contains a stereochemically specific hydrophobic triazole(Pro)-Trp cluster, with a short N-terminal peptide extension providing groups for potential main chain and side chain hydrogen bonding. The results of this work argue that the pharmacophore for dual antagonism is structurally limited, thereby enhancing the potential to develop minimized peptidomimetic HIV-1 entry inhibitors that simultaneously suppress binding of envelope protein to both of its host cell receptors. The results also argue that the target epitope on gp120 is relatively small, pointing to a localized allosteric inhibition site in the HIV-1 envelope that could be targeted for small-molecule inhibitor discovery.


Assuntos
Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Peptídeos/metabolismo , Triazóis/metabolismo , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Antígenos CD4/metabolismo , Domínio Catalítico , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Dados de Sequência Molecular , Peptídeos/química , Ressonância de Plasmônio de Superfície , Termodinâmica , Triazóis/química
2.
J Virol ; 81(7): 3645-8, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17251295

RESUMO

The AIDS epidemic continues to spread at an alarming rate worldwide, especially in developing countries. One approach to solving this problem is the generation of anti-human immunodeficiency virus (HIV) compounds with inhibition spectra broad enough to include globally prevailing forms of the virus. We have examined the HIV type 1 (HIV-1) envelope specificity of a recently identified entry inhibitor candidate, HNG-105, using surface plasmon resonance spectroscopy and pseudovirus inhibition assays. The combined results suggest that the HNG-105 molecule may be effective across the HIV-1 subtypes, and they highlight its potential as a lead for developing therapeutic and microbicidal agents to help combat the spread of AIDS.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Internalização do Vírus/efeitos dos fármacos , Estrutura Molecular , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
3.
Biochemistry ; 45(38): 11401-13, 2006 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-16981700

RESUMO

The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O2, CO, and CN-, among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine, whereas, in myoglobins, it is a serine involved in an intricate hydrogen-bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala --> Ser replacement. The substitution resulted in minor structural perturbations, but the response of the holoprotein to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary-structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased 2-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.


Assuntos
Alanina/química , Substituição de Aminoácidos , Globinas/química , Globinas/metabolismo , Serina/química , Synechocystis/química , Sequência de Aminoácidos , Cianetos/metabolismo , Compostos Férricos/química , Compostos Férricos/metabolismo , Heme/química , Heme/metabolismo , Histidina/química , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mioglobina/química , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Cachalote , Relação Estrutura-Atividade , Temperatura , Ureia/metabolismo
4.
Micron ; 35(1-2): 71-2, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15036295

RESUMO

The truncated hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 ligate the heme iron with two axial histidines (HisF8 and HisE10). In addition, these two proteins are able to form a heme-protein cross-link between a vinyl substituent and a histidine at position 16 of the H helix. The product is a protein with improved resistance to thermal and acid denaturation.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Hemoglobinas/química , Hemoglobinas Truncadas
5.
J Biol Inorg Chem ; 9(2): 183-94, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14727166

RESUMO

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the (1)H, (15)N, and (13)C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N(2)-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal p K(a) whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.


Assuntos
Cianobactérias/metabolismo , Heme/química , Hemoglobinas/metabolismo , Histidina/química , Ácidos , Substituição de Aminoácidos , Reagentes de Ligações Cruzadas , Elétrons , Temperatura Alta , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Plasmídeos , Conformação Proteica , Desnaturação Proteica
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